Abstract: The aim of this study was to construct a recombinant eukaryotic plasmid coexpressing GP3, GP5 and M proteins of porcine reproductive and respiratory syndrome virus (PRRSV). GP3, GP5 and M protein gene fragments of PRRSV LN strain were amplified by PCR and cloned into vector pcDNA3.1(+) to produce the recombinant plasmid pcDNA3.1GP3GP5M. The plasmid was transfected into COS7 cells, and immunological responses to the plasmid were investigated in BALB/c mice and piglets. The recombinant plasmid pcDNA3.1GP3GP5M was transfected into COS7 cells, PCR identification and indirect immunofluorescence assay proved that GP3, GP5M gene of PRRSV were expressed in COS7 cells. The result of Western blotting showed that the GP3 and GP5 and M proteins were coexpressed and formed fusion protein. The BALB/c mice were injected with recombinant plasmid pcDNA3.1GP3GP5M to evaluate the induced immunological responses in vivo. The specific detectable antiPRRSV neutralizing antibodies were produced in the vaccinated mice at 2 weeks and reached a peak 1:32 at 8 weeks after primary vaccination. In addition, it was observed that the 1:41:8 neutralizing antibodies of the vaccinated piglets. The results showed that the recombinant plasmid pcDNA3.1GP3GP5M has been constructed successfully, and the recombinant plasmid has well immunity, the recombinant plasmid could be a basis to eukaryotic vector vaccine for PRRSV infection.